The objective of the proposed research is the isolation of the gene coding for a subunit of DNA-dependent RNA polymerase II from eukaryotic cells. The subunit is that which binds the mushroom toxin Alpha-amanitin causing inhibition of the activity of RNA polymerase II. Studies on cell lines resistant to the toxicity of Alpha-amanitin (ama) have suggested that the control of the synthesis of the subunit may involve autogenous regulation. The isolated gene will be used to study this regulation of transcription or translation in vitro. The methods to be used for the gene isolation is based on the ligation of fragmented DNA from CHO-amar cells to the dual vector thymidine kinase (tk+) pBR322 and selection for the phenotype tk+, ama after transformation of tk- cells. The DNA from these cells will be used for transformation and subsequent selection of the gene in E. coli using the phenotype of the plasmid pBR322. After characterization of the isolated gene as coding for the subunit of RNA polymerase II, the gene can be used to study transcription and translation in vitro using available systems. The results of these studies can begin to reveal an understanding of mechanisms of gene expression unique to eukaryotes as well as provide information on the control of the level of cellular RNA polymerase II molecules.